Modified ribonucleic acids and uses thereof

Abstract

Disclosed herein is a modified ribonucleotide comprising a nucleoside comprising N4-acetylcytidine and/or 5-hydroxymethyluridine, and polyribonucleotides comprising the same. Also provided herein are compositions comprising a polyribonucleotide of the present disclosure and methods of making and using the same.

What is claimed is: 
     
       1. A polyribonucleotide encoding a payload comprising a polypeptide, wherein the polyribonucleotide comprises a modified ribonucleotide having a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has:
 (a) a 5′ monophosphate; 
 (b) a 5′ diphosphate; or 
 (c) a 5′ triphosphate and a structure of: 
 
       
         
           
           
               
               
           
         
         wherein the polyribonucleotide comprises cytidine residues and at least 25% of the cytidine residues comprise N4-acetylcytidine. 
       
     
     
       2. The polyribonucleotide of  claim 1 , wherein:
 less than 100% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. 
 
     
     
       3. The polyribonucleotide of  claim 1 , wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than N4-acetylcytidine. 
     
     
       4. The polyribonucleotide of  claim 3 , wherein the one or more modified ribonucleotides is 5-hydroxymethyluridine, and the modified ribonucleotide has:
 (a) a 5′ monophosphate; 
 (b) a 5′ diphosphate; or 
 (c) a 5′ triphosphate and a structure of: 
 
       
         
           
           
               
               
           
         
       
     
     
       5. The polyribonucleotide of  claim 1 , characterized in that when assessed in a cell, tissue or an organism that has been administered the polyribonucleotide:
 (a) reduced immunogenicity is observed relative to an appropriate reference comparator, 
 (b) increased cell viability is observed relative to an appropriate reference comparator, or 
 (c) both (a) and (b). 
 
     
     
       6. A polyribonucleotide encoding a payload comprising a polypeptide, wherein the polyribonucleotide comprises a modified ribonucleotide having a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and the modified ribonucleotide has:
 (a) a 5′ monophosphate; 
 (b) a 5′ diphosphate; or 
 (c) a 5′ triphosphate and a structure of: 
 
       
         
           
           
               
               
           
         
         wherein the polyribonucleotide comprises uridine residues and at least 25% of the uridine residues comprise 5-hydroxymethyluridine. 
       
     
     
       7. The polyribonucleotide of  claim 6 , wherein
 (i) less than 100% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine; 
 (ii) more than 60% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine; or 
 (iii) a combination thereof. 
 
     
     
       8. The polyribonucleotide of  claim 6 , wherein the polyribonucleotide further comprises one or more modified ribonucleotides other than 5-hydroxymethyluridine. 
     
     
       9. The polyribonucleotide of  claim 8 , wherein the one or more modified ribonucleotides is N4-acetylcytidine and the modified ribonucleotide has:
 (a) a 5′ monophosphate; 
 (b) a 5′ diphosphate; or 
 (c) a 5′ triphosphate and a structure of: 
 
       
         
           
           
               
               
           
         
       
     
     
       10. The polyribonucleotide of  claim 6 , characterized in that when assessed in a cell, tissue, or organism that has been administered the polyribonucleotide:
 (a) reduced immunogenicity is observed relative to an appropriate reference comparator, 
 (b)increased cell viability is observed relative to an appropriate reference comparator, 
 (c) increased expression of the payload is observed relative to an appropriate reference comparator, or 
 (d) a combination of (a)-(c). 
 
     
     
       11. The polyribonucleotide of  claim 10 , wherein reduced immunogenicity activation of an immune response comprises:
 (i) reduced activation of pathways of NFkb, IRF, or other cytokines resulting from inflammation in the cell, tissue or organism; or 
 (ii) reduced comprises reduced detection of uncapped RNA by a molecular sensor, wherein the molecular sensor is or comprises RIG-I; 
 (iii) both (i) and (ii). 
 
     
     
       12. The polyribonucleotide of  10 , wherein reduced immunogenicity allows for:
 (i) repeated dosing of the polyribonucleotide; 
 (ii) administration of a higher dose of the polyribonucleotide as compared to an appropriate reference comparator; or 
 (iii) both (i) and (ii). 
 
     
     
       13. The polyribonucleotide of  claim 12 , wherein the reference comparator comprises an otherwise similar cell, tissue or organism that has been administered a comparable polyribonucleotide that includes:
 (i) fewer N4-acetylcytidine nucleosides; 
 (ii) fewer 5-hydroxymethyluridine nucleosides; or 
 (iii) or both (i) and (ii). 
 
     
     
       14. The polyribonucleotide of  claim 10 , wherein the increase in expression of the payload is about 1.2-fold to about 20 compared to the reference comparator. 
     
     
       15. A composition comprising the polyribonucleotide of  claim 1 . 
     
     
       16. The composition of  claim 15 , wherein the composition is a pharmaceutical composition comprising an immunogenic composition; a vaccine, a gene therapy, a chemotherapy, a protein replacement therapy, an immunotherapy, a cell engineering therapy, or a combination thereof. 
     
     
       17. A cell comprising a polyribonucleotide according to  claim 1 . 
     
     
       18. A composition comprising the polyribonucleotide of  claim 6 . 
     
     
       19. The composition of  claim 18 , wherein the composition is a pharmaceutical composition comprising an immunogenic composition; a vaccine, a gene therapy, a chemotherapy, a protein replacement therapy, an immunotherapy, a cell engineering therapy, or a combination thereof. 
     
     
       20. A cell comprising a polyribonucleotide according to  claim 6 . 
     
     
       21. A polyribonucleotide encoding a payload comprising a polypeptide, wherein the polyribonucleotide comprises a modified ribonucleotide comprising a nucleoside comprising:
 (I) a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and the modified ribonucleotide has: (a) a 5′ monophosphate; (b) a 5′ diphosphate; or (c) a 5′ triphosphate and a structure of: 
 
       
         
           
           
               
               
           
         
       
       and
 (II) an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has:(a) a 5′ monophosphate; (b) a 5′ diphosphate; or (c) a 5′ triphosphate and a structure of: 
 
       
         
           
           
               
               
           
         
       
       wherein the polyribonucleotide comprises:
 (i) cytidine residues and at least 25% of the cytidine residues comprise N4-acetylcytidine; and 
 (ii) uridine residues and at least 25% the uridine residues comprise 5-hydroxymethyluridine. 
 
     
     
       22. The polyribonucleotide of  claim 21 , wherein:
 (a) the polyribonucleotide comprises cytidine residues and at least 50% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; 
 (b) the polyribonucleotide comprises uridine residues and at least 50% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine; or 
 (c) both (a) and (b). 
 
     
     
       23. The polyribonucleotide of  claim 21 , characterized in that when assessed in a cell, tissue, or organism that has been administered the polyribonucleotide:
 (a) reduced immunogenicity is observed relative to an appropriate reference comparator, 
 (b)increased cell viability is observed relative to an appropriate reference comparator, 
 (c) increased expression of the payload is observed relative to an appropriate reference comparator, or 
 (d) a combination of (a)-(c). 
 
     
     
       24. The polyribonucleotide of  claim 1 , wherein at least 50% of the cytidine residues comprise N4-acetylcytidine. 
     
     
       25. The polyribonucleotide of  claim 1 , wherein at least 75% of the cytidine residues comprise N4-acetylcytidine. 
     
     
       26. The polyribonucleotide of  claim 6 , wherein at least 50% of the uridine residues comprise 5-hydroxymethyluridine. 
     
     
       27. The polyribonucleotide of  claim 6 , wherein at least 75% of the uridine residues comprise 5-hydroxymethyluridine. 
     
     
       28. The polyribonucleotide of  claim 21 , wherein:
 (a) the polyribonucleotide comprises cytidine residues and at least 90% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine; 
 (b) the polyribonucleotide comprises uridine residues and at least 90% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine; or 
 (c) both (a) and (b). 
 
     
     
       29. The polyribonucleotide of  claim 21 , wherein 100% of the cytidine residues comprise N4-acetylcytidine and 100% of the uridine residues comprise 5-hydroxymethyluridine. 
     
     
       30. A composition comprising the polyribonucleotide of  claim 21 , wherein the composition is a pharmaceutical composition comprising an immunogenic composition; a vaccine, a gene therapy, a chemotherapy, a protein replacement therapy, an immunotherapy, a cell engineering therapy, or a combination thereof.